Dry Powder Fibrin Sealant

ABSTRACT

The invention provides a composition comprising a mixture of first microparticles that comprise fibrinogen and trehalose, and second microparticles that comprise thrombin and trehalose. The invention further provides methods for treating wounds by administering the novel microparticle composition.

CROSS-REFERENCE TO A RELATED APPLICATION

This application claims the benefit of U.S. provisional application Ser.No. 61/122,063, filed Dec. 12, 2008, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to a dry powder fibrin sealant.

BACKGROUND OF THE INVENTION

WO97/44015 describes a dry powder fibrin sealant based onmicro-particles of fibrinogen and thrombin. This has been demonstratedto be an easy-to-use, stable and efficacious topical haemostat. Theproduct can be used immediately, without reconstitution. On contact withaqueous fluid such as blood, the exposed active thrombin immediatelyconverts the exposed fibrinogen into insoluble fibrin polymers.

SUMMARY OF THE INVENTION

The novel fibrin sealant is a blend of spray-dried fibrinogen andthrombin, each of which has been individually co-spray dried with anexcipient. A number of excipients have been used in the fibrin sealantformulation to stabilise the active ingredients fibrinogen and thrombinand the physical stability evaluated. In addition, the fibrin sealantformulation has been exposed to electron beam/gamma irradiation or heatsterilisation in order to terminally sterilize the product. The resultsof the evaluation indicate that trehalose is the most effectiveexcipient in terms of protein protection during stability storage andelectron beam exposure. The superior stabilization afforded by thetrehalose-based formulations may be attributed to the higher glasstransition temperature of trehalose compared to other excipients such assucrose.

The influence of different parameters on the efficacy of the product wasdetermined in pig liver biopsy models and pig liver resection models.The efficacy of the fibrin sealant powder to stop severely bleedinginjuries, with blood loss of >10 ml/min, was enhanced by the opportunityto apply pressure directly after administration of the product. Fibrinsealant powders with a fibrinogen content of at least 4% w/w and athrombin content of at least 139 IU/g were shown to be effective instopping severe bleeding. The optimum fibrinogen and thrombin contentwas ˜7.5% w/w and ˜400 IU/g, respectively. Fibrinogen and thrombin from3 different suppliers all performed equally well, demonstrating therobustness of the product. Terminal sterilization of the product usingelectron beam or gamma irradiation of up to 15 or 25 kGy had no effecton the efficacy of the product and is considered to reduce the risk ofbacterial contamination before use. In summary, the invention provides afibrin sealant product that demonstrates high efficacy at low fibrinogenlevels in severely bleeding wounds and can be terminally sterilizedusing standard irradiation methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of the particle size distribution of spray-driedthrombin:trehalose according to the invention.

FIG. 2 is a plot of the particle size distribution of spray-driedfibrinogen:trehalose according to the invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

Respective fibrinogen-containing and thrombin-containing solublemicroparticles can be formulated together, in stable, dry form. Thisformulation can be subsequently activated, as desired, to give a fibrinsealant that is useful in wound therapy and surgical repair. It can meetthe primary objectives of achieving good flow properties, enhanced,effective delivery to the active site, and dissolution only at the site,not in the delivery system.

The content of fibrinogen in the microparticles containing it may beabout 0.1 to 50% w/w, preferably about 0.5 to 20 w/w. The content ofthrombin in the microparticles containing it may be about 10 to 20,000IU/g, preferably about 25 to 1,000 IU/g.

Microparticles comprising fibrinogen or thrombin may be prepared by theprocedures described in WO92/18164, WO96/09814 and WO96/18388. Thesespray-drying and associated particle manipulation processes enable theproduction of soluble protein microcapsules with defined sizedistribution, e.g. of up to 50 μm in diameter. For example, as describedin those documents, the microparticles may be produced reproducibly,e.g. with 90% or more (by volume) up to 30 μm, e.g. 10 to 20 μm, insize.

Microparticles of the invention are preferably prepared by spray-drying.Typically, a 2-fluid nozzle is used which utilises compressed air duringthe atomisation process; this results in the production of hollowmicroparticles. The maximum particle size (X50) of microparticles thatcan be manufactured using this atomisation system on the Niro MobileMinor spray dryer is ˜30 μm. Preferred X50 values for the micoparticlesof the invention are between 5 and 50 microns, most preferably between10 and 20 microns.

Microparticles of the invention may be prepared by spray-drying asolution of the active component with trehalose alone. An alternativeprocedure comprises co-spray-drying, in which fibrinogen or thrombin andanother wall-forming material are formulated and spray-dried, to givemicroparticles in which the active component is incorporated in the wallof the particle. The product is preferably amorphous or in the form of aglass, as measured by a suitable technique such as FTIR or DSC., with aglass transition temperature of at least 50 Celsius, most preferably atleast 80 Celsius.

The fibrinogen or thrombin may be full-length or any active fragmentthereof. Fragments are known; see Caller et al, J. Clin. Invest.89:546-555 (1992). Fibrinogen raw material may be a frozen solution,although, lyophilised powder which requires reconstitution prior tospray-drying may be used.

Suitable other proteins may be naturally occurring or recombinant. Theymay act as “wall-forming materials”, as described in WO92/18164, wherevarious examples are given. A preferred material is HSA (human serumalbumin). For example, fibrinogen is spray-dried alone or in thepresence of varying amounts of excipients such as HSA (e.g. fibrinogen:HSA ratios of 1:1, 1:3, 3:1) and trehalose. Other suitable substitutesfor HSA include surfactants, such as Tween 20, Tween 80, Poloxamer 407or Poloxamer 188.

Calcium ion, e.g. as calcium chloride, may be incorporated in thethrombin feedstock. Alternatively, calcium chloride may be added to themicrocapsules after processing.

Microparticles of the invention may be sterilised, if necessary ordesired. Sterile processing, electron beam irradiation, y-irradiationand ethylene oxide are examples of suitable techniques.

Although the components of the microcapsules in a fibrin sealant of theinvention are preferably water-soluble, and the microparticles arepreferably obtained by spray-drying a suitable solution, themicroparticles that are obtainable may be free-flowing, discrete andsubstantially anhydrous, with a residual moisture content preferably nogreater than 5% w/w, most preferably no greater than 3% w/w. This meansthat the compounds of fibrin sealant in accordance with this inventionare not activated until they are wetted, e.g. by coming into contactwith liquid at a wound site. The active components may therefore bedelivered as a dry mixture, although separate application of thedifferent microparticles is also envisaged.

A dry powder fibrin sealant product may be of particular value whereapplication to a large surface area is required. This includes surgeryand repair of traumatic injuries to various organs such as the liver andspleen. A further advantageous application is in skin grafting for burnspatients, and specifically where skin epidermal sheets are cultured invitro and then transferred to the wound site. The use of fibrin sealantin the latter indication may be particularly effective in patients withextensive burns, providing a biocompatible anchorage for skin grafts. Itmay also be suitable in the treatment of topical ulcers.

The following Examples illustrate the invention.

Example 1

Spray-dried fibrinogen microparticles were prepared by dissolving 73.8 ghuman fibrinogen in 1650 mL water containing 275.1 g trehalosedihydrate. The resultant solution was spray-dried on a Niro Mobile Minorspray dryer using the following operating parameters:

Inlet temperature: 160° C. Atomisation type: 2 - Fluid Nozzle Liquidinsert: 0.5 mm Atomisation pressure: 0.5 bar Feed rate: 18 g/minute

The spray-dried powder had a particle size (X50, geometric diameter) of18.4 μm and a fibrinogen content of 152 mg/g. The moisture content(Karl-Fischer) was 2%.

Spray-dried thrombin microparticles were prepared by dissolving 751,230IU human thrombin in 1653 mL water containing 11.5 g calcium chloridedihydrate and 507.3 g trehalose dihydrate. The resultant solution wasspray-dried on a Niro Mobile Minor spray dryer using the followingoperating parameters:

Inlet temperature: 160° C. Atomisation type: 2 - Fluid Nozzle Liquidinsert: 0.5 mm Atomisation pressure: 0.5 bar Feed rate: 18 g/minute

The spray-dried powder had a particle size (X50, geometric diameter) of12.5 μm and a thrombin content of 977 IU/g. The moisture content(Karl-Fischer) was 3%.

The two spray-dried powders were blended in a 1:1% w/w ratio using adrum mixer at 18 rpm for 15 minutes. The resultant blend had a particlesize of 15.5 μm, and a fibrinogen content of 69.1 mg/g.

The respective particle size distributions are shown in FIGS. 1 and 2.

FIG. 1 shows the cumulative distribution as follows:

x₀/μm Q₃/% 1.80 6.71 2.20 8.45 2.60 10.02 3.00 11.48 3.60 13.58 4.4016.36 5.20 19.25 6.20 23.15 7.40 28.33 8.60 33.97 10.00 40.87 12.0050.65 15.00 64.07 18.00 75.17 21.00 83.61 25.00 91.27 30.00 96.53 36.0099.11 42.00 99.85 50.00 100.00 60.00 100.00 72.00 100.00 86.00 100.00102.00 100.00 122.00 100.00 146.00 100.00 174.00 100.00 206.00 100.00246.00 100.00 294.00 100.00 350.00 100.00

Evaluation: WINDOX 5.1.2.0, HRLD Product: Fibrocaps Revalidation:Density: 1.00 g/cm³, shape factor: 1.00 Reference measurement: Disp.Meth: Set up for Fibrocaps R/M 08-20 11:42:05 C_(opt) = 1.56%Contamination: 0.00%

Trigger condition: Fibrocaps User parameters: Time base: 200.00 ms BatchNumber: PV Thrombin Start: c.opt >= 0.2% Formulation: EM/08/126 Valid:always Name: aks Stop: 2.000 s c.opt <= 0.2% or Run Number: Run 1 10000s real timeFIG. 2 shows the cumulative distribution as follows:

x₀/μm Q₃/% 1.80 2.68 2.20 3.58 2.60 4.53 3.00 5.52 3.60 7.06 4.40 9.195.20 11.39 6.20 14.20 7.40 17.63 8.60 21.10 10.00 25.15 12.00 30.8815.00 39.17 18.00 46.85 21.00 53.70 25.00 61.53 30.00 69.48 36.00 76.9142.00 82.47 50.00 87.80 60.00 92.11 72.00 95.07 86.00 96.87 102.00 97.94122.00 98.72 146.00 99.34 174.00 99.81 206.00 100.00 246.00 100.00294.00 100.00 350.00 100.00

Evaluation: WINDOX 5.1.2.0, HRLD Product: Fibrocaps Revalidation:Density: 1.00 g/cm³, shape factor: 1.00 Reference measurement: Disp.Meth: Set up for Fibrocaps R/M 08-29 13:35:41 C_(opt) = 7.30%Contamination: 0.00%

Trigger condition: Fibrocaps User parameters: Time base: 200.00 ms P1:SD Fibrinogen: trehalose clinical Start: c.opt >= 0.2% P2: EM/08/129Valid: always P3: TR Stop: 2.000 s c.opt <= 0.2% or P4: run 1 10000 sreal time

Example 2

Four batches of microparticles were produced, using the followingformulations and a Mini spray dryer,

-   -   200 mg/ml trehalose-200 units/ml thrombin    -   200 mg/ml sucrose-200 units/ml thrombin-1% HSA w-v    -   200 mg/ml trehalose-40 mg/fibrinogen    -   200 mg/ml sucrose-40 mg/ml fibrinogen.

The spray-drying parameters were selected so as to produce particles inthe region of 10 μm.

Thrombin Formulation:

Inlet temperature: 130° C. Outlet temperature: −80° C. AtomisationAirflow: 5/-min Drying Airflow: 5/-sec Feed Rate: 5.0 g-min

Fibrinogen Formulation:

Inlet temperature: 130° C. Outlet temperature: −83° C. AtomisationAirflow: 15/-min Drying Airflow: 5/-sec Feed Rate: 3.0 g-min

Each of the microcapsules batches was aliquoted into clear 10 ml glassvials both as separate components and as excipient-matched blends. Astability study at 4° C. was conducted over four weeks. Four timepointswere selected; initial, 1 week, 2 weeks and 4 weeks, and the followingassays were employed to compare the effect of the different excipientson the stability and bioactivity retention.

Fibrinogen Analysis used a polyconal antibody to human fibrinogen as acapture antibody and a second peroxidise-labelled antibody to humanfibrinogen is used for detection in a chromogenic assay.

Thrombin Analysis was based on a commercial substrate which is sensitiveto thrombin and gives a colour change which can be measured. The initialrate of change in absorbance is proportional to thrombin concentration.

Particle Size was measured using a LS230 Laser Sizer in conjunction withMedium Chain Trigylceride oil to determine the particle size of thespray dried material.

Thermogravimetric Analysis was carried out to assess moisture content.

Flow time was the time taken for a microcapsule blend to pass through afunnel of a pre-determined size was used as a comparative measure offlowability between batches.

Angle of Repose indicates the flowability of a powder and was measuredby the calculation of the angle created upon the flow of a powderthrough a funnel and subsequent accumulation on a flat surface.

Packed and tap density were measured using the Jolting Volume Meter andthe values used in Carrs Compressibility Index (% CCI).

Clot Strength utilises the formulation of a clot from a blend in aplastic syringe. A bead is suspended in the syringe prior to clotformation and the weight required to pull the bead through the clot isrecorded.

Adhesive Strength: blends are applied to a piece of rat skin via a 10 mlglass pipette fitted with a compressed air supply. The weight requiredto separate two pieces of the tissue bonded together by a blend is usedas a measurement of adhesive strength. (This assay is based on a Gottlobskin test method-Gesting and Lerner: Autologous fibrinogen for tissueadhesion haemostatis (1983)).

Additional assays were performed at the four week timepoint; SDS PAGE toassess the effects of spray-drying on the structural integrity of thebioactives and a BCA assay for total protein determination. Scanningelectron micrographs (SEM) were also obtained for each of the individualformulations.

Results demonstrated no significant changes over the stability periodfor either formulation, but the data do suggest a greater retention ofactivity generated for the trehalose formulation when compared with thesucrose formulation. The clot strength values also indicate an increasedactivity retention with the trehalose formulation. The addition of HSAto the trehalose-thrombin formulation showed no significant differencesin bioactivity retention compared to the trehalose-thrombin formulation.The flow properties were retained over the stability period which isreflected in the consistent adhesive strength values.

SDS PAGE data demonstrated the retention of structural integritypost-spray-drying.

Scanning electron micrographs revealed similar morphology for allformulations.

Dry heat viral inactivation step was conducted for 72 hours at 80° C.

The individual fibrinogen and thrombin components were assessed usingELISA and chromogenic assays respectively and the blends were analysedusing the clot strength assay. The results are documented in Table 1.

TABLE 1 Dry Heat Sterilisation Bioactivity - Clot Concentration per 100mg Strength Sample spray-dried Product (g) Trehalose - Thrombin 101.6units (97%) * microcapsules Trehalose - Fibrinogen 14.17 mg (103%) *microcapsules Trehalose - Active blend * 64.8 g Sucrose - Thrombin  93.8units (89%) * microcapsules Sucrose - Fibrinogen 11.04 mg (80.5) *microcapsules Sucrose - Active blend * 62.7 gTheoretical thrombin concentration=105 units-100 mg spray-dried productTheoretical fibrinogen concentration=13.7 mg-100 mg spray-dried product% Retention is shown in bracketsExpected clot strength=˜70 g

The bioanalytical results indicate the excipient trehalose allows agreater retention of the active during the dry heat step.

Gamma-irradiation employed 25 KG at a rate of 8 KG-hour. Samples wereexposed to these irradiation conditions both as separate components andas a blend.

The components were assessed using ELISA and chromogenic assaysrespectively and the blends examined via the clot strength assay. Theresults are documented in Table 2.

TABLE 2 Gamma Irradiation Sterilisation Bioactivity - Clot Concentrationper 100 mg Strength Sample spray-dried Product (g) Trehalose - Thrombin  71 units (71%) * microcapsules Trehalose - Fibrinogen 10.3 mg (73%) *microcapsules Trehalose - Active blend * 59.5 g Sucrose - Thrombin   71units (71%) microcapsules Sucrose - Fibrinogen  7.7 mg (55.5 *microcapsules Sucrose - Active blend * 36.9 g% Retention is shown in brackets.

The conditions investigated in the terminal sterilisation study (72hours at 80° C.) suggest that trehalose offers a higher level ofprotection to the protein, as reflected by the activity retention. Thisobservation may also suggest a trehalose formulation may be capable ofroom temperature storage. Gamma irradiation of the sucrose formulationresulted in a 50% drop in fibrinogen activity. The trehalose formulationwas found to have a significantly higher fibrinogen activity retention(70%) as measured by the ELISA and clot strength assays.

1. A composition, comprising a mixture of first microparticles thatcomprise fibrinogen and trehalose, and second microparticles thatcomprise thrombin and trehalose.
 2. The composition, as claimed in claim1, wherein said first microparticles contain 0.5 to 20% w/w fibrinogen.3. The composition, as claimed in claim 1, wherein said secondmicroparticles comprise 10 to 20,000 IU/g thrombin.
 4. The composition,as claimed in claim 3, wherein said second microparticles comprise 25 to1,000 IU/g thrombin.
 5. A method of treating a wound, comprisingadministering to the wound an effective amount of the composition asdefined in claim
 1. 6. The method, as claimed in claim 5, whichadditionally comprises applying pressure immediately after saidadministering.